Antiactivators prevent self-sensing in Pseudomonas aeruginosa quorum sensing

Abstract

Quorum sensing is described as a widespread cell density-dependent signaling mechanism in bacteria. Groups of cells coordinate gene expression by secreting and responding to diffusible signal molecules. Theory, however, predicts that individual cells may short-circuit this mechanism by directly responding to the signals they produce irrespective of cell density. In this study, we characterize this self-sensing effect in the acyl-homoserine lactone quorum sensing system of Pseudomonas aeruginosa. We show that antiactivators, a set of proteins known to affect signal sensitivity, function to prevent self-sensing. Measuring quorum-sensing gene expression in individual cells at very low densities, we find that successive deletion of antiactivator genes qteE and qslA produces a bimodal response pattern, in which increasing proportions of constitutively induced cells coexist with uninduced cells. Comparing responses of signal-proficient and -deficient cells in cocultures, we find that signal-proficient cells show a much higher response in the antiactivator mutant background but not in the wild-type background. Our results experimentally demonstrate the antiactivator-dependent transition from group- to self-sensing in the quorum-sensing circuitry of P. aeruginosa. Taken together, these findings extend our understanding of the functional capacity of quorum sensing. They highlight the functional significance of antiactivators in the maintenance of group-level signaling and experimentally prove long-standing theoretical predictions.

Keywords: Pseudomonas aeruginosa; acyl-homoserine lactone; antiactivation; quorum sensing; self-sensing.

Fig. 1.

Effect of antiactivation on QS-controlled genes. Cell density-dependent expression of lasB′-gfp, PAAR4′-gfp, rsaL′-gfp, and lasI′-gfp in the P. aeruginosa WT (red), the qteE qslA antiactivator double mutant (blue), and the lasI signal synthesis mutant (gray), grown in LB monoculture in a plate reader (n = 3). GFP fluorescence levels were normalized to cell density, expressed as OD₆₀₀.

Fig. 2.

Flow cytometry analysis of WT and antiactivator mutant cell populations carrying a lasI′-gfp reporter. (A) Selected histograms showing fluorescence distributions before, during, and after induction (left to right). The number on the top right indicates cell density (OD₆₀₀). The arrow indicates the subpopulation in the on state (from about a quarter in the qslA qteE mutant to absent in the WT). (B) Median GFP intensity of cell populations during the entire culturing period vs. cell density. Median values were determined from the respective histograms. Individual biological replicates are shown (n = 3). (C) Fraction of induced cells vs. cell density. Cells with a fluorescence intensity greater than 10³ were considered to be ON. All replicate data were fit with a single curve, using a Hill-type sigmoidal function. The gray line in B and C indicates the lasI mutant baseline. (D) Cell-density values resulting in half-maximal lasI activation determined from a curve fit to the data in C. Error bars indicate SD. All values are significantly different from each other as determined by one-way ANOVA (P < 0.05).

Fig. 3.

AHL sensitivity of antiactivator mutants. (A) Relative GFP expression as a function of 3OC12-HSL concentration. Signal synthesis mutants were grown to saturation with increasing concentrations of exogenous, synthetic 3OC12-HSL signal (n = 3). The expression of the lasI′-gfp reporter construct in these strains was measured using a fluorescence plate reader. The data were fit with a Hill-type sigmoidal curve to determine the half-maximal induction of each strain. (B) Signal concentrations resulting in half-maximal induction. Error bars indicate SD. With one exception, all pairwise comparisons are significantly different from each other as determined by one-way ANOVA (*P < 0.05).

Fig. 4.

Antiactivator-dependent self-sensing in cocultures of signal producers and nonproducers. Individual P. aeruginosa strains carrying the lasI′-gfp reporter in cocultures are denoted as follows: Q⁺, antiactivator (qslA qteE)-proficient; Q⁻, antiactivator (qslA qteE)-deficient; I⁺, signal (lasI)-proficient; l⁻, signal (lasI)-deficient. (A) Diagram of experimental coculture design with expected outcomes. Only one of two possible red-tag combinations is shown. (B) Selected histograms showing the fluorescence distributions of the I⁺ and I⁻ subpopulations in cocultures before, during, and after induction (left to right). The number on the top right indicates cell density (OD₆₀₀). The subpopulation labeled with E2-Crimson is represented by blue histograms and the subpopulation labeled with DsRed-Express2 is represented by red histograms. (C) Median GFP intensity of subpopulations during the culturing period vs. the cell density of the I⁺ strain (n = 4; two for each of the two red-tag combinations). (D) Response ratios vs. the cell density of the I⁺ strain. (E) Response ratios vs. the median GFP intensity of the I⁺ strain. In C–E, reciprocal labeling of I⁺ and I⁻ cells by the respective red tags is indicated by squares vs. triangles. (F) Mean presaturation response ratios of the Q⁺ and Q⁻ strain pairs. This mean response ratio was determined by averaging the individual response ratios of all samples for which the median GFP intensity of the I⁺ strain was below 20,000. Error bars represent SD of the mean. P values were calculated using a one-sample Student’s t test to determine significant difference from 1 and using a two-sample Student’s t test to determine significant difference between two samples. The superscripts ⁺ and ^– for both Q and I genes were changed to + and – for small font sizes to improve legibility.